Targeted introduction of a diphtheria toxin resistant mutation into the chromosomal EF-2 locus of Pichia pastoris and expression of immunotoxin in the EF-2 mutants

In an attempt to increase the production of a diphtheria toxin (DT) based immunotoxin by Pichia pastoris, we have created DT-resistant mutants that contain a substitution of arginine for glycine at position 701 in elongation factor 2 (EF-2). To achieve this, we first cloned and characterized the EF-2 gene (PEF1), and then made a construct pBLURA-Delta5'mutEF-2 that efficiently introduces specific mutations into the chromosomal EF-2 gene in P. pastoris by in vivo homologous recombination. pBLURA-Delta5(')mutEF-2 contains a selection marker URA3 and a 5' truncated form of the P. pastoris PEF1 that had been modified in vitro to carry the nucleotide mutations for the Gly(701) to Arg transition. Unlike the non-mutated strains, the EF-2 mutants are resistant to high-level intracellular expression of DT A chain that can catalyze the ADP-ribosylation. When used to express the secreted bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G4S), the EF-2 mutant strains showed increased viability compared to the non-mutated strains. However, they did not show an advantage over the non-mutated expressing strain in the production of the immunotoxin. Western blotting analysis revealed that although the EF-2 mutants did not increase the accumulation of intact A-dmDT390-bisFv(G4S) in the culture medium, they generated larger amounts of degraded products found in both the medium and cell pellets compared to the non-mutant expressing clone. In addition, double copy expression resulted in greater amounts of intact immunotoxin being retained within cellular compartments as well as degraded products. Based on these findings, we suggest that the secretory capacity may be rate limiting for divalent immunotoxin production in P. pastoris.     

In vitro succinylcholine hydrolysis in plasma of the African elephant (Loxodonta africana) and impala (Aepyceros melampus)

In elephants the time lapsed from i.m. injection of an overdose of the muscle relaxant succinylcholine (SuCh) until death, is significantly longer than in impala. To determine a difference in the rate of SuCh hydrolysis, once the drug enters the circulation, contributes to this phenomenon we have measured the rate of hydrolysis of SuCh in elephant and impala plasma, and by elephant erythrocytes. Rate of hydrolysis was determined by incubating SuCh in plasma or erythrocyte lysate at 37 degrees C and quantifying the choline produced. Plasma SuCh hydrolytic activity in elephant plasma (12.1+/-1.7 Ul(-1) mean+/-S.D.; n=9) was significantly higher than it was in impala plasma (6.6+/-0.6 Ul(-1); n=5), but were approximately 12 and 21 times lower, respectively, than in human plasma. Elephant erythrocyte lysate had no SuCh hydrolytic activity. Applying this data to previous studies, we can show that the ratio of SuCh absorption to SuCh hydrolysis is expected to be 1.25:1 and 1.41:1 for elephants and impala respectively. It will thus take at least 1.7 times longer for elephant to achieve a plasma SuCh concentration similar to that in impala. We conclude that a more rapid hydrolysis of SuCh in elephant plasma is one factor that contributes to the longer time to death compared to impala.     

Homocysteine accelerates endothelial cell senescence

In this study we demonstrate that exposure of cultured endothelial cells to homocysteine significantly accelerates the rate of endothelial senescence. Examination of telomere length demonstrates that homocysteine increases the amount of telomere length lost per population doubling. The effects of homocysteine on both senescence and telomere length are inhibited by treatment with the peroxide scavenger catalase. Chronic exposure of endothelial cells to homocysteine also increases the expression of two surface molecules linked to vascular disease, intracellular adhesion molecule-1 (ICAM-1) and plasminogen activator inhibitor-1 (PAI-1). Interestingly, the level of expression of both ICAM-1 and PAI-1 correlates with the degree of endothelial senescence. Taken together, these results suggest that homocysteine accelerates the rate of cellular senescence through a redox-dependent pathway. In addition, it suggests that chronic oxidative stress in the vessel wall may hasten the rate of senescence and that the senescent endothelial cell may in turn be pro-atherogenic.     

Endothelin-converting enzyme in human tissues

Our aim was to determine whether the expression of endothelin-converting enzyme in human tissues would correlate with the distribution of its substrate, big endothelin-1, and its product, the mature peptide. Site-directed antisera raised against the conserved C-terminus of the mammalian enzyme were used to measure the immunoreactive enzyme in microsomal fractions prepared from tissue homogenates and to localize staining to the endothelial cells lining large conduit and smaller resistance vessels within cardiac, adrenal, respiratory and brain tissue. The activity of endothelin-converting enzyme was measured and characterized in isolated endothelial cells. This pattern of staining in the vascular endothelium paralleled that of mature endothelin and big endothelin-1, and these peptides were detectable by radioimmunoassay in all tissues examined. Immunoreactive endothelin-converting enzyme localized to other cell types, including bronchial epithelial cells, and to fibres within the glial limitans, neuronal processes and cell bodies of the cerebral cortex. Although perivascular astrocytes in the subcortical white matter displayed intense endothelin-converting enzyme-like immunoreactivity, endothelin staining was not detected. The results suggest that endothelin-converting enzyme has a ubiquitous distribution within the human vascular endothelium and is positioned to catalyse the conversion of big endothelin-1 to the biologically active endothelin-1, which on release may contribute to the maintenance of basal tone in humans. Endothelin-converting enzyme localized to epithelial cells in peripheral tissues or astrocytes within the brain may be upregulated in pathophysiological conditions in which endothelin levels are increased and could represent a further target for therapeutic intervention by enzyme inhibitors. © 1998 Chapman & Hall     

Seasonal Patterns of Hormones,Macroparasites, and Microparasites in Wild African Ungulates: The Interplay among Stress,Reproduction, and Disease

Sex hormones, reproductive status, and pathogen load all affect stress. Together with stress, these factors can modulate the immune system and affect disease incidence. Thus, it is important to concurrently measure these factors, along with their seasonal fluctuations, to better understand their complex interactions. Using steroid hormone metabolites from fecal samples, we examined seasonal correlations among zebra and springbok stress, reproduction, gastrointestinal (GI) parasite infections, and anthrax infection signatures in zebra and springbok in Etosha National Park (ENP), Namibia, and found strong seasonal effects. Infection intensities of all three GI macroparasites examined (strongyle helminths, Strongyloides helminths, and Eimeria coccidia) were highest in the wet season, concurrent with the timing of anthrax outbreaks. Parasites also declined with increased acquired immune responses. We found hormonal evidence that both mares and ewes are overwhelmingly seasonal breeders in ENP, and that reproductive hormones are correlated with immunosuppression and higher susceptibility to GI parasite infections. Stress hormones largely peak in the dry season, particularly in zebra, when parasite infection intensities are lowest, and are most strongly correlated with host mid-gestation rather than with parasite infection intensity. Given the evidence that GI parasites can cause host pathology, immunomodulation, and immunosuppression, their persistence in ENP hosts without inducing chronic stress responses supports the hypothesis that hosts are tolerant of their parasites. Such tolerance would help to explain the ubiquity of these organisms in ENP herbivores, even in the face of their potential immunomodulatory trade-offs with anti-anthrax immunity.     

A Phase I Trial of DFMO Targeting Polyamine Addiction in Patients with Relapsed/Refractory Neuroblastoma

BackgroundNeuroblastoma (NB) is the most common cancer in infancy and most frequent cause of death from extracranial solid tumors in children. Ornithine decarboxylase (ODC) expression is an independent indicator of poor prognosis in NB patients. This study investigated safety, response, pharmacokinetics, genetic and metabolic factors associated with ODC in a clinical trial of the ODC inhibitor difluoromethylornithine (DFMO) ± etoposide for patients with relapsed or refractory NB.ConclusionsDFMO doses of 500-1500mg/m2/day are safe and well tolerated in children with relapsed NB. Children with the minor T allele at rs2302616 of the ODC gene with relapsed or refractory NB had higher levels of urinary polyamine markers and responded better to therapy containing DFMO, compared to those with the major G allele at this locus. These findings suggest that this patient subset may display dependence on polyamines and be uniquely susceptible to therapies targeting this pathway.

Trial Registration

Clinicaltrials.gov NCT#01059071     

Gender-specific fatty acid profiles in platelet phosphatidyl-choline and -ethanolamine

Previous studies suggested that women synthesise docosahexaenoic acid (DHA) more efficiently from their precursors than men. This study investigated the relationship between diet, platelet phospholipids fatty acids and gender. Dietary intake and platelet phosphatidyl-choline (PC) and phosphatidylethanolamine (PE) fatty acids were determined in Caucasian 40 men and 34 women. Absolute and %energy intakes of arachidonic acid (AA), eicosapentaenoic acid (EPA), and DHA, and the ratios of total n-6/n-3 PUFA and linoleic/alpha-linolenic acids did not differ between the sexes. However, women had higher DHA in PC (1.19 vs 1.05 wt%, p<0.05) and PE (3.62 vs 3.21 wt%, p<0.05) than men. Also EPA (1.10 vs 0.93 wt%, p<0.05) was higher in women's PE. Conversely, men had elevated AA and total n-6 fatty acids in PC. The higher platelet DHA levels and lower platelet AA/EPA and AA/DHA ratios in women of child-bearing age compared with men, may lead to less platelet aggregation and vaso-occlusion.     

Effects of Trp- and Arg-Containing Antimicrobial-Peptide Structure on Inhibition of Escherichia coli Planktonic Growth and Biofilm Formation

Biofilms are sessile microbial communities that cause serious chronic infections with high morbidity and mortality. In order to develop more effective approaches for biofilm control, a series of linear cationic antimicrobial peptides (AMPs) with various arginine (Arg or R) and tryptophan (Trp or W) repeats [(RW)_n-NH₂, where n = 2, 3, or 4] were rigorously compared to correlate their structures with antimicrobial activities affecting the planktonic growth and biofilm formation of Escherichia coli. The chain length of AMPs appears to be important for inhibition of bacterial planktonic growth, since the hexameric and octameric peptides significantly inhibited E. coli growth, while tetrameric peptide did not cause noticeable inhibition. In addition, all AMPs except the tetrameric peptide significantly reduced E. coli biofilm surface coverage and the viability of biofilm cells, when added at inoculation. In addition to inhibition of biofilm formation, significant killing of biofilm cells was observed after a 3-hour treatment of preformed biofilms with hexameric peptide. Interestingly, treatment with the octameric peptide caused significant biofilm dispersion without apparent killing of biofilm cells that remained on the surface; e.g., the surface coverage was reduced by 91.5 ± 3.5% by 200 μM octameric peptide. The detached biofilm cells, however, were effectively killed by this peptide. Overall, these results suggest that hexameric and octameric peptides are potent inhibitors of both bacterial planktonic growth and biofilm formation, while the octameric peptide can also disperse existing biofilms and kill the detached cells. These results are helpful for designing novel biofilm inhibitors and developing more effective therapeutic methods.Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics (5). Native AMPs are part of the host defense in organisms ranging from bacteria to insects, plants, and animals (14). They are capable of eliminating a broad spectrum of microorganisms, including viruses, bacteria, and fungi (4, 14). Compared with widespread antibiotic resistance (38), resistance to AMPs is rare, possibly because AMPs directly target cell membranes that are essential to microbes (14, 29). In addition, no cross-resistance has been observed in clinic due to the diversity of peptide sequences (42). Thus, native and synthetic AMPs offer potential alternatives to antibiotics for treating drug-resistant infections (3, 26, 27).In mammalian innate immune systems, some AMPs are produced constitutively, while others are inducible within hours after detection of invading microorganisms (4, 13). Although the detailed mechanism of AMPs'' activities remains elusive (5), AMPs are known to disrupt cell membranes of microbes, interfere with metabolism, and/or target cytoplasmic components (41). Most known AMPs are cationic and amphiphilic (29). It is hypothesized that the initial interaction occurs via an electrostatic attraction between the AMP molecule and microbial membrane. Cationic AMPs can cover bacterial membranes, disrupt the membrane potential, create pores across the membrane, and consequently cause the leak of cell contents and cell death (27, 41). AMPs are relatively selective in targeting microbes rather than mammalian cells, most likely because of the fundamental differences between microbial and host membranes (41), e.g., a higher abundance of negatively charged phospholipids and an absence of cholesterol in microbial membranes.Known AMPs vary dramatically in sequence, size (from 12 to 50 amino acids), and structure (α-helices or β-sheets) (23). However, most AMPs have two types of side chains with relatively conservative sequences: positively charged basic residues, containing arginine (R), lysine (K), and/or histidine (H), that presumably mediate the interaction with the negatively charged microbial membrane, and bulky hydrophobic residues, rich in tryptophan (W), proline (P), and/or phenylalanine (F), that facilitate permeabilization and membrane disruption (26).Although AMPs are promising agents for antimicrobial therapies (15), only a few have made it to clinical trials and applications, with varied success (15, 42). There are several issues that need further development. First, the MICs of AMPs are relatively high compared to those of conventional antibiotics. Recent studies suggest that the peptide/lipid (P/L) ratio needs to be higher than a threshold to allow the AMPs to be oriented perpendicular to the membrane so that pores can be created to kill bacteria (22, 30). Thus, an optimization of peptide structure and size may improve their antimicrobial activities. In addition to the high MICs, the wide application of AMPs is also hindered by their high manufacturing costs and the cytotoxicity of some AMPs.Given the limit of currently available AMPs, it is important to develop more effective AMPs with reduced manufacturing cost and enhanced activity (17, 26, 28, 39). Strøm et al. (39) chemically synthesized a series of short cationic AMPs containing repeating R and W residues in order to identify the minimal pharmacophore with high antimicrobial activities. The data suggest that tetrapeptides or capped tripeptides are effective and there is no correlation between the order of amino acids and antimicrobial activity. Liu et al. (26) analyzed the effects of chain length on the activities of AMPs with repeating pharmacophore sequences (RW)_n-NH₂ (n = 1, 2, 3, 4, or 5). The tests of antimicrobial activities and the hemolysis of red blood cells suggest that (RW)₃-NH₂ has the optimal chain length. Although longer chains are more potent antimicrobials, they can stimulate hemolysis.Most of the AMP studies to date are focused on planktonic bacteria. However, the majority of pathogenic bacteria tend to adhere to surfaces and form sessile microbial communities with highly hydrated structures of secreted polysaccharide matrix, collectively known as biofilms (9). Biofilms can tolerate up to 1,000 times more antibiotics and disinfectants than their planktonic counterparts (2, 7, 8). For example, Folkesson et al. (12) reported that biofilm formation of E. coli K-12 increases its tolerance to polymyxin E, a polypeptide antibiotic that kills Gram-negative bacteria by disrupting membranes (34, 40). Since biofilms are involved in 80% of human bacterial infections (1), it is necessary to study biofilm inhibition and dispersion by AMPs.In this study, a series of linear peptides (RW)_n-NH₂ (where n = 2, 3, or 4) were studied for the effects of their activities on planktonic cells and biofilms of E. coli to understand the structural effects on the antimicrobial activities of AMPs. We chose E. coli RP437 in this study because it is one of the model strains for biofilm research and allows us to compare the data with those of our previous studies (6, 16, 19, 20).     

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

Introduction

Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear.

Methods

This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mM glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined.

Results

TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used.

Conclusions

Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.